Editorial. Platelet purinergic receptors and non-thrombotic diseases.Simon C. Pitchford1* and Dingxin Pan.11Pulmonary Pharmacology Unit, Institute of Pharmaceutical Science, King’s College London, London, UK.*Author for correspondence and reprint requests:Dr Simon PitchfordPulmonary Pharmacology UnitInstitute of Pharmaceutical Science5.43 Franklin Wilkins Building150 Stamford StreetWaterloo CampusKing’s College LondonLondon UKSE1 9NHPhone: +44 2078484266Fax: +44 207 8484788Simon.pitchford@kcl.ac.uk

Editorial on the themed issue:

Biased signaling is a natural result of GPCR allosteric function and should be expected from any and all synthetic agonists. Therefore, it may be encountered in all agonist discovery projects and must be considered as a beneficial (or possible detrimental) feature of new candidate molecules. While bias is easily detected , the synoptic nature of GPCR signaling makes translation of simple in vitro bias to complex in vivo systems problematic. The practical outcome of this is a difficulty in predicting the therapeutic value of biased signaling due to the failure of translation of identified biased signaling to in vivo agonism. This is discussed in this review as well as some new ways forward to improve this translation process and better exploit this powerful pharmacologic activity.

Background and Purpose: P2X4 receptors (P2X4R) are ligand gated cation channels that are activated by extracellular adenosine 5′-triphosphate (ATP) released by neurons and glia. The receptors are widely expressed in the brain and have fractional calcium currents comparable to NMDA receptors. Although P2X4Rs were described to modulate synaptic transmission and plasticity, their involvement in shaping neuronal network activity remains to be elucidated. Exp. Approach: We investigated the effects of P2X receptors on network and synaptic level using local field potential electrophysiology, whole cell patch clamp recordings and calcium imaging in fast spiking parvalbumin positive interneurons (PVINs) in rat and mice hippocampal slices. The stable ATP analogue ATPγS, selective antagonists and P2X4R knockout mice were used. Key results: The P2XR agonist ATPγS reversibly decreased the power of gamma oscillations. This inhibition could be antagonized by the selective P2X4R antagonist PSB-12062 and was not observed in P2X4-/- mice. The phasic excitatory inputs of CA3 PVINs were one of the main regulators of the gamma power. Associational fibre compound excitatory postsynaptic currents (cEPSCs) in CA3 PVINs were inhibited by P2X4R activation. This effect was reversible, dependent on intracellular calcium and dynamin-dependent internalization of AMPA receptors. Conclusions and Implications: The results indicate that P2X4Rs are an important source of dendritic calcium in CA3 PVINs, thereby regulating excitatory synaptic inputs onto the cells and the state of gamma oscillations in the hippocampus. P2X4Rs represent an effective target to modulate hippocampal network activity in pathophysiological conditions such as Alzheimer’s disease and schizophrenia.

Chronic Obstructive Pulmonary Disease (COPD) is a major incurable health burden, ranking as the 3rd leading cause of death worldwide, mainly driven by cigarette smoking. COPD is characterised by persistent airway inflammation, lung function decline, and premature aging with the presence of pulmonary senescent cells. This review proposes that cellular senescence, a state of stable cell cycle arrest linked to ageing; induced by inflammation and oxidative stress in COPD, extends beyond the lungs and impacts the systemic circulation. This “spill over” of senescent cells contributes to brain inflammation and damage, increasing the risk of neurological comorbidities, such as stroke, cerebral small vessel disease, and Alzheimer’s disease. The review explores the role of cellular senescence in COPD-associated brain conditions and investigates the relationship between cellular senescence and circadian rhythm in COPD. Additionally, it discusses potential therapies, including senomorphic and senolytic treatments, as novel strategies to halt or improve COPD progression.

The KEAP1-NRF2 system plays a central role in cytoprotection and defense mechanisms against oxidative stress. Because KEAP1 serves as a biosensor for electrophiles by using its reactive thiols and because NRF2 is a transcriptional factor regulating genes involved in the sulfur-mediated redox reactions, the KEAP1-NRF2 system has been regarded as a sulfur-utilizing cytoprotective mechanism. NRF2 is a key regulator of cytoprotective genes, such as antioxidant and detoxification genes, and also to possess potent anti-inflammatory activity. NRF2 has been recently focused as a great modifier/regulator for the cellular metabolism and mitochondrial function. Particularly, the NRF2-mediated regulatory mechanisms of metabolites and mitochondria has been considered diverse, but has not been fully-clarified yet. This review article provides an overview of the molecular mechanisms that regulate NRF2 signaling and its cytoprotective roles, and also highlights NRF2 contribution to the cellular metabolism, particularly in the context of mitochondrial function and newly found sulfur metabolism.

Background and Purpose: 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the oxoreduction of cortisone to cortisol, thereby amplifying levels of active glucocorticoids. It is considered a pharmaceutical target in metabolic disease and cognitive impairments. 11β-HSD1 also converts some 7oxo-steroids to their 7β-hydroxy forms. A recent study in mice described the ratio of tauroursodeoxycholic acid (TUDCA)/tauro-7oxolithocholic acid (T7oxoLCA) as a biomarker for decreased 11β-HSD1 oxoreductase activity. The present study aimed to evaluate the equivalent bile acid ratio glycoursodeoxycholic acid (GUDCA)/glyco-7oxolithocholic acid (G7oxoLCA) as a biomarker for pharmacological 11β-HSD1 inhibition in humans and compare it with the currently applied urinary (5α-tetrahydrocortisol+tetrahydrocortisol)/tetrahydrocortisone ((5αTHF+THF)/THE) ratio. Experimental Approach: Bile acid profiles were analyzed by ultra-HPLC tandem-MS in blood samples from two independent, double-blind placebo-controlled clinical studies on the orally administered selective 11β-HSD1 inhibitor AZD4017. The blood GUDCA/G7oxoLCA ratio was compared with the urinary tetrahydro-glucocorticoid ratio for the ability to detect 11β-HSD1 inhibition. Key Results: No significant alterations were observed in the bile acid profiles following 11β-HSD1 inhibition by AZD4017, except for an increase of the secondary bile acid G7oxoLCA. The enzyme product/substrate ratio GUDCA/G7oxoLCA was found to be more reliable to detect 11β-HSD1 inhibition than the absolute G7oxoLCA concentration in both cohorts. Comparison of the blood GUDCA/G7oxoLCA ratio with the urinary (5αTHF+THF)/THE ratio revealed that both ratios successfully detect 11β-HSD1 inhibition. Conclusion and Implications: 11β-HSD1 inhibition does not cause major alterations in bile acid homeostasis. The GUDCA/G7oxoLCA ratio represents the first blood biomarker of pharmacological 11β-HSD1 inhibition and may replace or complement the urinary (5αTHF+THF)/THE ratio biomarker.

Background and Purpose: Peripheral nerve trauma-induced dysregulation of pain-associated genes in the primary sensory neurons of dorsal root ganglion (DRG) contributes to neuropathic pain genesis. RNA-binding proteins participate in gene transcription. We hypothesized that RALY, an RNA-binding protein, participated in nerve trauma-induced dysregulation of DRG pain-associated genes and nociceptive hypersensitivity. Methods and results: Immunohistochemistry staining showed that RALY was expressed exclusively in the nuclei of DRG neurons. Peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve produced time-dependent increases in the levels of Raly mRNA and RALY protein in injured DRG. Blocking this increase through DRG microinjection of adeno-associated virus 5 (AAV5)-expressing Raly shRNA reduced the CCI-induced elevation in the amount of eukaryotic initiation factor 4 gamma 2 (eIF4G2) mRNA and eIF4G2 protein in injured DRG and mitigated the development and maintenance of CCI-induced nociceptive hypersensitivity, without altering basal (acute) response to noxious stimuli and locomotor activity. Mimicking DRG increased RALY through DRG microinjection of AAV5 expressing Raly mRNA upregulated the expression of eIF4G2 mRNA and eIF4G2 protein in the DRG and led to hypersensitive responses to noxious stimuli in the absence of nerve trauma. Mechanistically, CCI promoted the binding of RALY to the promoter of eIF4G2 gene and triggered its transcriptional activity. Conclusion and Implications: Our findings indicate that RALY participates in nerve trauma-induced nociceptive hypersensitivity likely through transcriptionally triggering eIF4G2 expression in the DRG. RALY may be a potential target in neuropathic pain management.

Background and Purpose Development of core concepts in disciplines such as biochemistry, microbiology, and physiology transformed teaching. They provided the foundation for the development of teaching resources for global educators, as well as valid and reliable approaches to assessment. An international research consensus recently identified 25 core concepts of pharmacology. The current study aimed to define and unpack these concepts. Experimental approach A two-phase, iterative approach, involving 60 international pharmacology education experts was used. The first phase involved drafting definitions for the core concepts and identifying key sub-concepts via a series of online meetings and asynchronous work. These were refined in the second phase, through a two-day hybrid workshop followed by a further series of online meetings and asynchronous work. Key Results The project produced consensus definitions for a final list of 24 core concepts and 103 sub-concepts of pharmacology. The iterative, discursive methodology resulted in the modification concepts from the original study, including the change of ‘drug-receptor interaction’ to ‘drug-target interaction’ and the change of the core concept ‘agonists and antagonists’ to sub-concepts of drug-target interaction. Conclusion and Implications The definitions and sub-concepts of the 24 core concepts provide an evidence-based foundation for pharmacology curricula development and evaluation. The next steps for this project include the development of a concept inventory to assess acquisition of the concepts, as well as the development of cases studies and educational resources to support teaching by the global pharmacology community, and student learning of the most critical and fundamental concepts of the discipline.

Background and Purpose: Sepsis-surviving adult individuals commonly develop immunosuppression and increased susceptibility to secondary infections, outcome mediated by the axis IL-33/ILC2s/M2 macrophages/Tregs. Nonetheless, the long-term immune consequences of pediatric sepsis are indeterminate. We sought to investigate the role of age in the genesis of immunosuppression following sepsis. Experimental Approach: Here, we compared the frequency of Tregs, the activation of the IL33/ILC2s axis in M2 macrophages, and the DNA methylation of epithelial lung cells from post-septic infant and adult mice. Likewise, sepsis-surviving mice were inoculated intranasally with Pseudomonas aeruginosa or by subcutaneous inoculation of the B16 melanoma cell line. Finally, blood samples from sepsis-surviving patients were collected and the concentrations of IL-33 and Tregs frequency were assessed. Key Results: In contrast to 6-week-old, 2-week-old mice were resistant to secondary infection and did not show impairment in tumour controls upon melanoma challenge. Mechanistically, increased IL-33 levels, Tregs expansion, and activation of ILC2s and M2-macrophages were observed in 6-week-old but not 2-week-old post-septic mice. Moreover, impaired IL-33 production in 2-week-old post-septic mice was associated with increased DNA methylation in lung epithelial cells. Notably, IL-33 treatment boosted the expansion of Tregs and induced immunosuppression in 2-week-old mice. Clinically, adults but not pediatric post-septic patients exhibited higher counts of Tregs and sera IL-33 levels. Conclusion and Implications: These findings demonstrate a crucial and age-dependent role for IL-33 in post-sepsis immunosuppression. Thus, a better understanding of this process could lead to differential treatments for adult and pediatric sepsis.

Autism spectrum disorders (ASD) are diagnosed in 1/100 childbirth worldwide, based on two core symptoms, deficits in social interaction and communication and stereotyped behaviours. G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors that mediate the transfer of extracellular signals to convergent intracellular signalling and downstream cellular responses that are dysregulated in ASD. Despite hundreds of GPCRs are expressed in the brain, only 23 GPCRs are genetically associated to ASD according to the Simons Foundation Autism Research Initiative (SFARI) gene database: oxytocin OTR, vasopressin V1A, V1B, metabotropic glutamate mGlu5, mGlu7, GABAB, dopamine D1, D2, D3, serotoninergic 5-HT1B, β2-adrenoceptor, cholinergic M3, adenosine A2A, A3, angiotensin AT2, cannabinoid CB1, chemokine CX3CR1, orphan GPR37, GPR85 and olfactory OR1C1, OR2M4, OR2T10, OR52M1. Here, we review the therapeutical potential of these 23 GPCRs, in addition to 5-HT2A, 5-HT6 and 5-HT7 for their relevance to ASD. We discuss their genetic association with ASD, the effects of their genetic and pharmacological manipulation in animal models and humans, their existing pharmacopeia towards core symptoms of ASD and rank them based on these evidences. Among these 23 GPCRs, we highlight that OTR, V1A, mGlu5, D2, 5-HT2A, CB1, and GPR37 are the best therapeutic targets. We conclude that the dysregulation of GPCRs and their signalling is a convergent pathological mechanism of ASD and their therapeutic potential has only begun as multiple GPCRs could mitigate ASD.

In recent years increasing evidence suggests that commensal microbiota may play an important role in health and disease, including cerebrovascular disease. Gut microbes impact physiology, at least in part by metabolizing dietary factors and also host-derived substrates and then generating active compounds including toxins. The purpose of the current review is to highlight the complex interplay between microbiota, their metabolites and essential functions for human health ranging from regulation of the metabolism and the immune system to modulation of brain development and function. We also discuss the role of gut dysbiosis in cerebrovascular disease, specifically in acute and chronic stroke phases and the possible implication of intestinal microbiota in post-stroke cognitive impairment and dementia, and we identify potential therapeutic opportunities of targeting microbiota in this context.

The central nervous system (CNS) has long been considered an immune-privileged site, with minimal interaction between immune cells, particularly of the adaptive immune system. Previously, the presence of immune cells in this organ was primarily linked to events involving disruption of the blood-brain barrier (BBB) or inflammation. However, current research has shown that immune cells are found patrolling CNS under homeostatic conditions. Specifically, T cells of the adaptive immune system are able to cross the BBB and are associated with aging and cognitive impairment. In addition, T-cell infiltration has been observed in pathological conditions, where inflammation correlates with poor prognosis. Despite ongoing research, the role of this population in the aging brain under both physiological and pathological conditions is not yet fully understood. In this review, we provide an overview of the interactions between T cells and other immune and CNS parenchymal cells, and examine the molecular mechanisms by which these interactions may contribute to normal brain function and the scenarios in which disruption of these connections lead to cognitive impairment. A comprehensive understanding of the role of T cells in the aging brain and the underlying molecular pathways under normal conditions could pave the way for new research to better understand brain disorders.

Background and Purpose: Blood-brain barrier (BBB) breakdown is one of the most crucial pathological changes of cerebral ischemia-reperfusion (I/R) injury. Trilobatin (TLB), a naturally occurring food additive, exerts neuroprotective effect against cerebral I/R injury as demonstrated in our previous study. This study was designed to investigate the effect of TLB on disruption of BBB after cerebral I/R injury. Experimental Approach: Rats with focal cerebral ischemia caused by transient middle cerebral artery occlusion (MCAO) and brain microvascular endothelial cells along with human astrocytes to mimic blood brain barrier (BBB) injury caused by oxygen and glucose deprivation (OGD) followed by reoxygenation (OGD/R). Key results: The results showed that TLB effectively maintained the integrity of BBB and inhibited neuronal loss following cerebral I/R challenge. Furthermore, TLB dramatically increased tight junction proteins including ZO-1, occludin and claudin 5, as well as decreased the levels of apolipoprotein E (APOE) 4, cyclophilin A (CypA), and phosphorylated nuclear factor kappa B (NF-κB), thereby reduced proinflammatory cytokines. In addition, TLB also decreased Bax/Bcl-2 ratio and cleaved-caspase 3 level along with reduced the number of apoptotic neurons. Intriguingly, molecular docking and transcriptomics predicted MMP9 was a prominent gene evoked by TLB treatment. Furthermore, the protective effect of TLB on OGD/R-induced the loss of BBB integrity in human brain microvascular endothelial cell and astrocyte co-cultures in vitro was markedly reinforced by knockdown of MMP9. Conclusions and implications: Our findings reveal a novel property of TLB: saving BBB disruption following cerebral I/R via targeting MMP9 and inhibiting APOE4/CypA/NF-κB axis.

Background and Purpose: Myocardial infarction (MI) is the leading cause of mortality globally due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we demonstrated that overexpression of 4 cell cycle factors, CDK1, CDK4, cyclin B1, and cyclin D1 (4F), induced cell division in ~20% of the post-mitotic CMs overexpressed 4F. The current study aims to identify a small molecule that augments 4F-induced CM cycle induction. Experimental Approach, Key Results: Screening of small molecules with a potential to augment 4F-induced cell-cycle induction in 60-day-old mature human induced pluripotent cardiomyocytes (hiPS-CMs) revealed N-(4,6-Dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide (NDPPC), which activates cell cycle progression in 4F-transduced hiPS-CMs. Autodock tool and Autodock vina computational methods showed that NDPPC has a potential interaction with the binding site at the human p38⍺ mitogen-activated protein kinase (p38⍺ MAP kinase), a critical negative regulator of the mammalian cell cycle. A p38⍺ MAP kinase activity assay showed that NDPPC inhibits its activity in a dose-dependent manner. Overexpression of p38⍺ MAP kinase in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect. Conclusion and Implications: NDPPC is a novel inhibitor for p38⍺ MAP kinase and is a promising drug to augment CM cell cycle response to the 4F. NDPPC could become an adjunct treatment with other cell cycle activators for heart failure treatment.

GPR84 is an understudied rhodopsin-like class A G protein-coupled receptor which is arousing particular interest from a therapeutic perspective. Not least this reflects that gpr84 expression is significantly up-regulated following acute inflammatory stimuli and in inflammatory diseases and that receptor activation plays a role in regulating pro-inflammatory responses and migration of cells of the innate immune system such as neutrophils, monocytes, macrophages and microglia. Although most physiological responses of GPR84 reflect receptor coupling to Gαi/o G-proteins, several studies indicate that agonist-activated GPR84 can also recruit arrestin adaptor proteins and this regulates receptor internalisation and desensitisation. To date, very little is known on the patterns of GPR84 phosphorylation and how these might control these processes. Here, we consider what is known on the molecular basis of GPR84 signalling with a focus on how GRK-mediated phosphorylation regulates arrestin protein recruitment and receptor function.

Dopamine transmission in the striatum is a critical mediator of the rewarding and reinforcing effects of commonly misused psychoactive drugs. G protein-coupled receptors (GPCRs) that bind a variety of neuromodulators including dopamine, endocannabinoids, acetylcholine, and endogenous opioid peptides regulate dopamine release by acting on several components of dopaminergic circuitry. Striatal dopamine release can be driven by both somatic action potential firing and local mechanisms that depend on acetylcholine released from striatal cholinergic interneurons. GPCRs that primarily regulate somatic firing of dopamine neurons via direct effects or modulation of synaptic inputs are likely to impact distinct aspects of behavior and psychoactive drug actions compared with GPCRs that primarily regulate local acetylcholine-dependent dopamine release in striatal regions. This review will highlight mechanisms by which GPCRs modulate dopaminergic transmission and the relevance of these findings to psychoactive drugs involved in substance use disorders.

Background and Purpose: The ionotropic purinergic trimeric receptor P2X3 is a new drug target other than the opioid receptor for the treatment of refractory chronic cough (RCC). However, the only marketed P2X3 antagonist, Gefapixant/AF-219, has a side effect of taste disorders due to simultaneous action on the human P2X2/3 (hP2X2/3) heterotrimer. Therefore, selective molecules with high affinity for the hP2X3 homotrimer and low affinity for the hP2X2/3 heterotrimer have potential in iteration 2.0 RCC drug development, such as Sivopixant/S-600918, a clinical phase II RCC candidate with lower taste disturbance than Gefapixant. S-600918 and its analogue (3-(4-((3-chloro-4-isopropoxyphenyl)amino)-3-(4-methylbenzyl)-2,6-dioxo-3,6-dihydro-1,3,5-triazin-1(2H)-yl)propanoic acid (DDTPA) exhibit both high affinity and high selectivity for hP2X3 homotrimers compared to hP2X2/3 heterotrimer. The mechanism of its druggable site and this high selectivity is not clear. Experimental Approach: Here, we reveal a novel allosteric mechanism that distinguishes this drug candidate from other P2X3 inhibitors through chimera construction, site covalent occupation, metadynamics, mutagenesis, and electrophysiology. Key Results: We suggest that the tri-symmetric site adjacent to the upper vestibule determines the high affinity and selectivity of S-600918/DDTPA for hP2X3. Only four amino acids of the hP2X2 upper body domain swapped with hP2X3, allow the hP2X2/3 heterotrimer to gain comparable affinity for S-600918/DDTPA as the hP2X3 homotrimer. Conclusion and Implications: Thus, we have revealed the molecular basis for the cough suppressive effects and reduced side effects of new RCC clinical candidates from the perspective of receptor-ligand recognition, which may provide information critical for the development of new drugs targeting P2X3 for indications such as RCC, idiopathic pulmonary fibrosis (IPF), and primary hypertension.